Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels.

Von Willebrand factor (VWF) is the carrier protein of the anti-haemophilic Factor VIII (FVIII) in plasma. It has been reported that the infusion of FVIII concentrate in haemophilia A patients results in lowered VWF plasma levels. However, the impact of F8-deficiency on VWF plasma levels in F8-/y mice is unresolved. In order to avoid confounding variables, we back-crossed F8-deficient mice onto a pure C57BL/6J background and analysed VWF plasma concentrations relative to C57BL/6J WT (F8+/y) littermate controls. F8-/y mice showed strongly elevated VWF plasma concentrations and signs of hepatic inflammation, as indicated by increased TNF-α, CD45, and TLR4 transcripts and by elevated macrophage counts in the liver. Furthermore, immunohistochemistry showed that expression of VWF antigen was significantly enhanced in the hepatic endothelium of F8-/y mice, most likely resulting from increased macrophage recruitment. There were no signs of liver damage, as judged by glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) in the plasma and no signs of systemic inflammation, as white blood cell subsets were unchanged. As expected, impaired haemostasis was reflected by joint bleeding, prolonged in vitro clotting time and decreased platelet-dependent thrombin generation. Our results point towards a novel role of FVIII, synthesized by the liver endothelium, in the control of hepatic low-grade inflammation and VWF plasma levels.

Gut microbiota regulate hepatic von Willebrand factor synthesis and arterial thrombus formation via Toll-like receptor-2.

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/- , and heterozygous Vwf+/- mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2-/- mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth.

Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs). Here, we report that colonization of germ-free (GF) Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R) showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

Recombinant streptavidin-C3bot for delivery of proteins into macrophages.

We demonstrated previously that monocytes and macrophages are target cells for the Rho-modifying Clostridium botulinum C3 ADP-ribosyltransferase. Here, we report the construction, expression and characterization of a recombinant streptavidin-C3 fusion protein which allows for delivery of biotin-labelled molecules into the cytosol of macrophages via enzymatically inactive C3bot1E174Q. The enzyme domain of diphtheria toxin was used as cargo to demonstrate proof of principle. This transport system could represent an attractive tool for experimental monocyte/macrophage pharmacology.

Streptavidin-conjugated C3 protein mediates the delivery of mono-biotinylated RNAse A into macrophages.

The C3 toxin produced by Clostridium botulinum (C3bot) catalyzes the mono-ADP-ribosylation of the small GTPases Rho A, B and C, resulting in their inactivation. Recently, a specific endocytotic uptake mechanism of C3bot was identified in macrophages and myeloid leukemia cells. Here, we present a novel delivery system based upon a mutant C3bot devoid of ADP-ribosylation activity (C3Mut) and wild-type streptavidin (Stv). The C3Mut moiety mediates endocytosis into macrophages, whereas Stv functions as an adaptor protein for attaching biotinylated molecules to facilitate their subsequent internalization. First, a bioconjugate consisting of recombinant C3Mut and Stv was generated via a thioether linkage that tightly interacted with biotinylated bovine serum albumin as demonstrated by dot blot analysis. We then showed the internalization of C3Mut-Stv into J774A.1 macrophages by confocal microscopy and observed translocation into the cytosol using cell fractionation. The C3Mut-Stv bioconjugate did not affect cell viability. Next, we prepared mono-biotinylated RNase A, which was attached to the C3Mut-Stv transporter, and demonstrated its C3Mut-Stv-mediated delivery into the cytosol of J774A.1 cells. Finally, C3Mut-Stv also promoted the efficient uptake of mono-biotinylated lysozyme into J774A.1 cells, highlighting its versatility. This study intriguingly demonstrates the use of the novel C3Mut-Stv delivery system for protein transduction and may provide a basis for future applications, in particular, of cytotoxic RNase A mutants.

Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen.

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF(N)DTA. LF(N) mediates uptake of LF(N)DTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF(N)DTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF(N)DTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF(N)DTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF(N)DTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF(N)DTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF(N)DTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin.

The C2 toxin produced by Clostridium botulinum is a binary AB-type exotoxin composed of the enzyme subunit C2I and the binding/translocation moiety C2II. After proteolytic activation, C2IIa mediates the subsequent internalization of C2I into the cytosol of mammalian target cells. The N-terminal domain of C2I (C2IN) is necessary for C2IIa-dependent uptake, but lacks the enzyme domain that is responsible for cytotoxicity. In the present study, we generated a delivery system building on C2IN and a truncated core streptavidin (Stv13) with enhanced solubility for the C2IIa-dependent internalization of biotinylated cargo molecules into mammalian cells. C2IN-Stv13 fusion protein expressed in Escherichia coli was obtained in high yields and purity. The affinity-purified protein formed tetramers and a defined higher order species in solution as shown by gel filtration and retained its biotin-binding properties, however with an obvious reduction in affinity. Uptake of C2IN-Stv13 into the cytosol of HeLa and other cancer cell lines was observed by immunoblot analysis, which was corroborated by confocal microscopy. In addition, the fusion protein was not cytotoxic and did not inhibit cell proliferation as determined by MTS assay. Finally, we demonstrated the C2IN-Stv13/C2IIa-mediated uptake of biocytin-Alexa 488 as cargo into HeLa cells, underscoring the functionality of the generated transport system.